Optimizing Archaeal Lipid Biosynthesis in Escherichia coli

Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.


Table of contents
Table S1: Corrected ion count Archaeal lipids and their precursors (Relates to figure 1B) Table S2: Ion counts PG species (relates to figure 1) Table S3: Ion counts PG species (relates to figure 1) Table S4: Corrected ion count Archaeal lipids and their precursors (Relates to figure 2) Table S5: Ion counts PG species (relates to figure 2) Table S6: Ion counts PE species (relates to figure 2) Table S7: List of Gro-APCL masses found in the E. coli mixed membrane lipidome Table S8: Bacterial strains used in this study Table S9: Plasmids used in the study Table S10: Primer sequences used in this study Table S11: Enzymes used for ether lipid production in E. coli Table S12: Enzymes used for isoprenoid overexpression

Figure S1 :
Figure S1: carS open reading frames (ORF) fix. Figure S2: Breakdown of ether lipid precursors in engineered E. coli strains.Figure S3: Ratios of bacterial, archaeal and hybrid cardiolipin species.Figure S4: Growth curves of EL+ strain treated with Cerulenin.

Figure S1 :
Figure S1: carS open reading frames (ORF) fix.The pAC09 vector from Caforio et al. 2018 has two start codons (orange box) close after the RBS (blue box) resulting in two open reading frames (arrow), one of which is out of frame.This issue is fixed in pMH09 and in all derivatives of pMH09.

Figure S2 :
Figure S2: Breakdown of ether lipid precursors in engineered E. coli strains.Archaeal lipid species in strains with chromosomally integrated MEP/DOXP operon (MD) or Isoprenoid utilization pathway (IUP) were analyzed by LC-MS, normalized for the internal standard DDM, and plotted.Bars represent the mean of the datapoints (MD samples N=5 IUP samples N=3).Ass+ indicated heterologous overexpression of M. maripaludis archaetidylserine synthase.In case of IUP samples isoprenol was added to the media.

Figure S3 :
Figure S3: Ratios of bacterial, archaeal and hybrid cardiolipin species.LC-MS analysis of lipid extracts from strains with the archaeal lipid biosynthetic pathway with and without overexpression of IDI from a plasmid grown for 16 hours in M9 with 10μM IPTG in the presence and absence of 25mM isoprenol.Figure shows the ratio's between Gro_DPCL, Gro-DACL and Gro_APCL lipid species, where 100% is the total ion count of all cardiolipin species quantified.Data reflect averages ± SD (N=3).

Figure S4 :
Figure S4: Growth curves of EL+ strain treated with Cerulenin.Twelve overnight cultures of the EL+ strain were diluted 1:100 in M9 media (A) and M9 + 100 μg/ml Cerulenin (B), then incubated in a plate reader at 37C with shaking and OD600 was measured every 10 minutes for 45 hours.Two out of twelve show signs of growth after approximately 20 hours.

Table S1 : Corrected ion count Archaeal lipids and their precursors
(Relates to figure1B) LC-MS analysis of lipid extracts from strains without (EL-) and with (EL+) archaeal ether lipid biosynthesis pathway, including PssA overexpression (EL+_PssA+) and Ass overexpression (EL+_Ass+) grown for 16 hours in LB with 10μM IPTG for induction of EL biosynthesis genes.Numbers given are the determined ion counts of indicated archaeal lipid species as well as precursors, divided by the ion count of the internal control (DDM).

Table S2 : Ion counts PG species
(relates to figure1) LC-MS analysis of lipid extracts from strains without (EL-) and with (EL+) archaeal ether lipid biosynthesis pathway, including PssA overexpression (EL+_PssA+) and Ass overexpression (EL+_Ass+) grown for 16 hours in LB with 10μM IPTG for induction of EL biosynthesis genes.Numbers given are the determined ion counts of indicated phosphatidyl glycerol species divided by the ion count of the internal control (DDM).

Table S3 :
: Ion counts PE species (relates to figure1) LC-MS analysis of lipid extracts from strains without (EL-) and with (EL+) archaeal ether lipid biosynthesis pathway, including PssA overexpression (EL+_PssA+) and Ass overexpression (EL+_Ass+) grown for 16 hours in LB with 10μM IPTG for induction of EL biosynthesis genes.Numbers given are the determined ion counts of indicated phosphatidyl ethanolamine species divided by the ion count of the internal control (DDM).

Table S4 : Corrected ion count Archaeal lipids and their precursors
(Relates to figure2)Ion counts from indicated archaeal lipid species divided by an internal control as determined by LC-MS analysis of lipid extracts.Strains all have the archaeal lipid biosynthetic pathway.When Ass is overexpressed this is indicated by Ass+.C indicates chromosomal expression of the IUP (ScCK, AtIPK, EcIDI) while P indicates plasmid based expression.Alternatively only IDI (IDI) or IUP version 2 (SmDAGK, MvIPK, EcIDI) are expressed from a plasmid (IDI_V2).All strains are grown for 16 hours in M9 with 10μM IPTG for induction of EL biosynthesis genes, when indicated 25mM isoprenol was added to the media (Iso).

Table S5 :
Ion counts PG species (relates to figure 2)Raw ion counts from indicated PG species as determined by LC-MS analysis of lipid extracts.Strains all have the archaeal lipid biosynthetic pathway.When Ass is overexpressed this is indicated by Ass+.C indicates chromosomal expression of the IUP (ScCK, AtIPK, EcIDI) while P indicates plasmid based expression.Alternatively only IDI (IDI) or IUP version 2 (SmDAGK, MvIPK, EcIDI) are expressed from a plasmid (IDI_V2).All strains are grown for 16 hours in M9 with 10μM IPTG for induction of EL biosynthesis genes, when indicated 25mM isoprenol was added to the media (Iso).